Contact inhibition is the ability of cells to inhibit motility and proliferation after forming cell-cell contacts. Our long-term goal is to understand the molecular mechanism underlying contact inhibition in normal epithelial cells. Epithelial cell-cell contacts are mainly formed by E-cadherin-based adherens junctions, which function as a focal point of many signaling pathways regulating various cellular behaviors, including most likely, contact inhibition. We recently reported that translocation of a protein complex to cell-cell contacts is pivotal to the establishment of contact inhibition. These complexes, defined here as "Pak:PIX complexes", minimally contain the Rac/cdc42 effector molecules p21-activated kinase 1 (Pak1) and the Rac/cdc42-guanine exchange factor betaPIX. In subconfluent cells, Pak:PIX complexes are in focal contacts, where they regulate cell motility and morphology. How they are recruited to cell-cell contact sites and how this translocation can regulate proliferation, is however unknown. The objective of this proposal is to characterize the mechanisms by which Pak:PIX complexes translocate from focal contacts to cell-cell contacts when cells become confluent, and to determine how this affects mitogenic signaling through the MEK-ERK pathway. The central hypothesis is that, upon E-cadherin-mediated activation of phosphatidyl inositol-3-kinase, Pak:PIX complexes are recruited to cell-cell contacts. This translocation may uncouple mitogenic signaling. In aim 1, we will characterize the translocation of Pak:PIX complexes and will identify new binding partners, in aim 2, we will further define the role of E-cadherin, and in aim 3, we will investigate how Pak:PIX complexes control contact inhibition by regulating the mitogenic MEK-ERK signaling pathway. The proposed research is important, as it will help to understand how localized, subcellular dynamic regulation of Rho effector proteins integrates the many signals that cells receive from their extracellular environment. The experiments outlined in this proposal will specifically address the mechanism that regulates the cell density-dependent localization of Pak:PIX complexes, which is crucial for contact inhibition. Identification of the mechanism underlying contact inhibition will allow us to identify specific target molecules that can be employed for diagnostic purposes or drug design. We anticipate that the results of our studies will yield novel and fundamental insights into the biology of epithelial morphogenesis.